Anthrax is a disease caused by the sporulating bacteria Bacillus anthracis. Humans working with animal products are at risk for contracting anthrax, particularly individuals such as veterinarians, laboratory technicians, ranchers and employees working with the skin or hair of animals. Areas such as Iran, Turkey, Iraq, Pakistan, and sub-Saharan Africa are hyperendemic for anthrax, although the organism can be found in most areas of the world. General worldwide populations are vulnerable to B. anthracis infection as a consequence of acts of bioterror, whereas military troops are vulnerable to infection as a consequence of acts of bioterror and/or war.
Anthrax manifests disease in three different ways: inhalation anthrax disease, gastrointestinal anthrax disease, and cutaneous anthrax disease. Inhalation anthrax disease is caused by inhaling spores. Gastrointestinal anthrax is caused by ingesting spores in contaminated meat, and cutaneous anthrax disease occurs when spores contact an open wound. Untreated inhalation or gastrointestinal anthrax has a case fatality rate of essentially 100 percent while cutaneous anthrax has a case fatality rate of up to 25 percent.
For persons infected with anthrax, treatment success is limited by several factors, such as the increased incidence of antibiotic resistance, the overwhelming septic responses, which can occur before antibiotics could be effective, and treatment delays based upon decreased suspicion and poor methods of early detection. that all can lessen the chance of survival. It is known that early treatment of anthrax with antibiotics is essential to reduce mortality, although not completely effective with inhalation infections. Delays in treatment profoundly decrease survival rates. Early treatment, however, is difficult because initial symptoms of the infection resemble common, non-fatal infections. For example, the inhalation of anthrax spores may initially present symptoms resembling those of the common cold. In addition, symptoms of anthrax infection, depending on how the bacterium is contracted, may take seven to sixty days to appear. Moreover, even prompt, effective antibiotic treatment does not ameliorate the effects of circulating toxins, which can remain in the blood at high levels and continue to mediate their pathogenic effects.
The pathogenicity of Bacillus anthracis is expressed in two ways: a toxic effect made evident by the appearance of edema, and a so-called lethal toxic effect that may lead to the death of the subject infected. These effects are attributed to the presence of toxins produced by a combination of three proteins that represent the toxin system: protective antigen (PA), lethal factor (LF) and edema factor (EF). In humans and other mammals, toxins increase in the body even during early stages of infection when the host appears asymptomatic. This explains why delays in treatment can be fatal. Thus, there is not only a critical need for improved anthrax intervention therapies, including treatments that interfere with the deleterious actions of the toxins, but also a critical need for point-of-care, rapid, and extremely sensitive diagnostic tests to establish the presence of anthrax early in the infection.
Passive immunization, an effort to neutralize toxins with antibodies, usually polyclonal antibodies, has been used as a therapeutic intervention for a variety of bacterial infections (Keller and Stiehm, 2000). A major limitation of using polyclonal antisera in patients is the possibility of “serum sickness” due to a patient's immune response to proteins derived from a different species. In addition, higher affinity antibodies are more effective for toxin neutralization, but there is no general way to enhance intentionally the affinity of polyclonal sera or even monoclonal antibodies derived from hybridomas. In addition, traditional approaches using pooled donor intravenous immunoglobulins (IVIG) is not effective as normal donors do not have effective humoral immunity against Bacillus anthracis. 
Although vaccines against Bacillus anthracis are currently available, they are not optimal. Even the most recent commercially available vaccines require multiple primary injections, yearly boosters, and fail to provide proven long-term effectiveness against inhalation attacks and adverse events. In addition, currently available vaccines exhibit severe adverse effects.
The mechanism for anthrax toxicity is as follows. The 83 kDa form of protective antigen (PA83) is secreted from rapidly growing Bacillus anthracis cells and binds to a specific host cell surface receptor, such as TEM8 or CMG2. (H. M. Scobie and J. A. Young, Curr. Opin. Micro. 8, 106-112 (2005)). Subsequent cleavage by membrane-bound furin, and/or a furin-like protease, possibly PACE4, releases an amino terminal 20 kDa fragment, resulting in receptor-bound PA63. PA63 subunits then oligomerize into heptameric rings, which in turn create binding sites for the lethal factor and edema factor components of Bacillus anthracis toxins. Endocytosis of the receptor/toxin complex into acidic endosomes elicits a conformational change in PA63, whereby the A subunits (LF or EF) of the toxin are released into the endosome. Lysosomal acidification and subsequent receptor release facilitate irreversible membrane insertion of the oligomeric PA63 pore. The pore permits transport of LF and/or EF into the cytoplasm where they elicit their respective toxicities.
EF is a calcium/calmodulin-dependent adenylate cyclase that is toxic to most cell types and causes local inflammation and edema, but is not usually lethal. Edema toxin, composed of protective antigen and edema factor is been recently shown to cause tissue lesion and death in a mouse model (see “Bacillus anthracis edema toxin causes extensive tissue lesions and rapid lethality in mice” by Firoved, A. M., G. F. Miller, M. Moayeri, R. Kakkar, Y. Shen, J. F. Wiggins, E. M. McNally, W. J. Tang, and S. H. Leppla. Am J Pathol 167:1309-1320 (2005)).
LF is a cell-type specific metalloprotease that cleaves MAP-kinase-kinases and several peptide hormones. Lethal toxin, formed by the combination of protective antigen and lethal factor is a zinc dependent protease that results in lysis of macrophages. Lethal factor is the major virulence factor associated with anthrax toxicity and is thought to be responsible for systemic shock and death. Neither of the toxin A subunits are pathogenic in the absence of cytoplasmic delivery by PA or mechanical means (See, “Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process” by A. M. Friedlander J. Biol. Chem. 261, 7123 (1986)).
The crystal structures of PA83 and heptameric PA63 have been resolved (See, e.g., “Crystal-structure of the anthrax toxin protective antigen” by C. Petosa et al., Nature. 385, 833 (1997)). These structural data support the experimental data (See, e.g., “Characterization of lethal factor-binding and cell-receptor binding domains of protective antigen of Bacillus anthracis using monoclonal-antibodies” by S. F. Little et al., Microbiology-UK. 142, 707 (1996) and “The carboxyl-terminal end of protective antigen is required for receptor-binding and anthrax toxin activity” by Y. Singh et al., J. Biol. Chem. 266,15493 (1991)) that indicate that domain 4, the carboxy-terminus of PA63, is responsible for receptor-mediated uptake of the toxin complex.
Bacillus anthracis has been used for over sixty years as a biological weapon and is a likely agent for a large-scale attack based upon the relative ease of obtaining and growing the bacterium, as well as the stability of the spores. Weaponized anthrax was first developed in 1941 by the British government, which tested the release of anthrax spores on an island near Scotland (Mourez, M. Rev Physiol Biochem Pharmacol 152:135-164 (2004) and Greenfield, R. A et al. Am J Med Sci 323:299-315 (2002)). While no individuals were infected during this test, the island remained a biohazard for forty-five years until seawater and formaldehyde was used to sterilize the soil. Since that time, however, there have been accidental and deliberate releases of militarized anthrax resulting in human infections and deaths. An accidental release of anthrax from a USSR military facility in 1979 caused 68 deaths and resulted in infection of cattle up to 31 miles away. And in 2001, the deliberate spread of anthrax through infected letters in the US resulted in 22 cases of anthrax (11 cutaneous and 11 inhalation), with five deaths (see Guarner, J. et al. Am J Pathol 163:701-709 (2003) and Quinn, C. P. et al. J Infect Dis 190:1228-1236 (2004)). These incidents highlight the need for a safe, effective vaccine that provides protection from an aerosolized release of Bacillus anthracis spores and potential directed immunotherapeutics for early intervention.
The current US vaccine (anthrax vaccine absorbed, AVA) is an alhydrogel absorbed cell-free filtrate of the attenuated V770-NP1-R strain, a non-encapsulated bovine isolate. This vaccination is administered at 0, 2, 4 weeks and again at 6, 12, and 18 months with yearly boosters recommended. The primary data on this vaccine has been obtained from animal studies and indicates that, while AVA does not protect mice from lethal challenge with fully virulent strains of B. anthracis, vaccinated mice are protected against challenge with nonencapsulated strains. These models have also demonstrated that passive transfer of antibodies against the major toxin proteins (PA, LF, and EF) can provide protection against challenge with attenuated strains (see Little, S. F. et al. Infect Immun 65:5171-5175 (1997); Little, S. F. et al. Infect Immun 56:1807-1813 (1988); Price, B. M et al. Infect Immun 69:4509-4515 (2001) and Welkos, S., et al. Microbiology 147:1677-1685 (2001)). Antibodies to PA, LF, and EF have also been detected in serum samples of individuals diagnosed with clinical anthrax, but vaccination results in primarily anti-protective antigen antibodies. However, several studies using mice vaccinated with mutant strains of Bacillus anthracis have shown significant contributions of LF and EF to protection (see Mohamed, N. et al. Infect Immun 72:3276-3283 (2004) and Little, S. F. et al. Biochem Biophys Res Commun 199:676-682 (1994)). Thus enhancing the levels of antibodies against these proteins might enhance protection. Accordingly, because the current vaccine is not optimal, new and improved preventative compositions and methods are necessary.
What are needed are methods and compositions that can prevent, inhibit and diminish the symptoms of Bacillus anthracis infection. The compositions should be able to overcome the activity of anthrax toxins, namely protective antigen, lethal factor and edema factor. Preferably the compositions should be able to induce an immune response in an animal or human. Also needed are compositions that can be used to produce neutralizing antibodies directed to components of the anthrax toxins, such as lethal factor and edema factor. Furthermore, what are needed are methods and compositions that can be used for immunotherapy, specifically directed to Bacillus anthracis infection. Finally, the methods and compositions for preventing, inhibiting and diminishing the symptoms of Bacillus anthracis infection should preferably be non-toxic and produce few side effects.